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Highly efficient methods for reprogramming differentiated cells and for generating animals and embryonic stem cells from reprogrammed cells

By using a fertilized embryo as a recipient in the cloning process, the method enhances the efficiency of cloning and reduces ethical concerns, enabling the production of transgenic animals with specific traits and pluripotent stem cells without the need for human oocytes.

Active Publication Date: 2015-06-25
ADVANCED CELL TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach increases the efficiency of cloning and reduces the need for human oocytes, allowing for the production of transgenic animals with desired traits and ethical considerations by generating pluripotent stem cells and animals without the destruction of embryos, thereby overcoming the limitations of traditional breeding and SCNT methods.

Problems solved by technology

Traditional breeding processes are capable of producing animals with some specifically desired traits, but these traits are often accompanied by a number of undesired characteristics, and are often too time-consuming, costly and unreliable to develop.
Moreover, these processes are completely incapable of allowing a specific animal line from producing gene products, such as desirable protein therapeutics that are otherwise entirely absent from the genetic complement of the species in question (i.e., human or humanized plasma protein or other molecules in bovine milk).
An additional problem associated with existing stem cell technologies are the ethical considerations of using advanced human embryos to obtain stem cells.

Method used

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  • Highly efficient methods for reprogramming differentiated cells and for generating animals and embryonic stem cells from reprogrammed cells
  • Highly efficient methods for reprogramming differentiated cells and for generating animals and embryonic stem cells from reprogrammed cells
  • Highly efficient methods for reprogramming differentiated cells and for generating animals and embryonic stem cells from reprogrammed cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

The Effect of Lagging Time Between Nuclear Injection and Enucleation on Pronuclear (PN) Stage Zygotes

[0224]Nuclear injection was performed on sixty-four PN stage embryos. GFP positive mouse ES cell nuclei were transferred into PN stage zygotes. Embryos were then cultured 3, 6, 9, or 12 hours before enucleating the original pronucleus. The cloned embryos were next cultured and their development was observed. A high percentage of embryos at all time points reached the two-cell stage, but only embryos enucleated 3 hours after nuclear transfer reached the four-cell stage (see Table 2).

TABLE 2The effect of lagging time between nuclear injection andenucleation on PN stage mouse zygotes.Time afterTotal # ofinjection (hours)zygotes2C4C31911206191100914900129600

example 2

Serial Cloning Using PN Stage Zygotes and 2-Cell Stage Embryos

[0225]In an effort to attain further development of cloned embryos, serial cloning was performed. Nuclear injection was performed as described in Example 1. Embryos were then cultured 3 hours before enucleating the original pronucleus. The cloned embryos were next cultured until the 2-cell stage.

[0226]A transplantation of dissociated individual cloned embryo cells into normal fertilized 2-cell stage mouse embryos was done at 18 hrs after the first cloning. The recipient embryos were enucleated prior to the nuclear transfer. Individual cloned blastomere cells were transplanted into the perivitellin space of the enucleated 2-cell stage embryos. Electrofusion of the transplanted blastomere and the enucleated embryo was performed by giving a single pulse of 150V DC for 15 microseconds.

[0227]The serially cloned embryos were cultured in KSOM medium and monitored for further development. Two of six embryos developed into blastoc...

example 3

Somatic Cell Cloning Using Two Cell Stage Mouse Embryos

[0228]It was hypothesized that cloned blastomeres in a mosaic embryo might be stimulated to develop further by non-cloned cells. One of the two blastomeres of a 2-cell stage mouse embryo was enucleated, and immediately after enucleation, an ES cell nuclei was injected into the enucleated blastomere. Embryos were cultured without any further manipulation in KSOM.

[0229]The cloned blastomeres divided the next day and contributed to GFP positive cells forming mosaic embryos (FIG. 2A-C). When these embryos developed to the 8-cell stage at least 3 blastomeres originated from the cloned blastomeres (FIG. 2B). Four of these cloned embryos developed into blastocyts (see Table 4). GFP positive blastomeres integrated into part of the blastocysts.

TABLE 4The effect of helper cells inside of the same zona pellucidawith cloned blastomeres in 2-cell stage cloned embryos.Total ## survived8C-Blasto-of 2 cellsinjection4CMcyst14844

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Abstract

The present invention relates generally to the field of somatic cell nuclear transfer (SCNT) and to the creation of cloned animals and cells. The disclosure relates to a method of cloning a mammal, obtaining pluripotent cells such as embryonic stem cells, or for reprogramming a mammalian cell using an oocyte and a fertilized embryo.

Description

RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application Nos. 60 / 902,970 filed 23 Feb. 2007, 60 / 918,543 filed 16 Mar. 2007, 60 / 993,772 filed 14 Sep. 2007 and 61 / 009,432 filed 28 Dec. 2007, the contents of which are incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention relates generally to the field of somatic cell nuclear transfer (SCNT) and to the generation of animals and cells.BACKGROUND OF THE INVENTION[0003]Advances in stem cell technology, such as the isolation and propagation in vitro of human embryonic stem cells (“hES” cells), constitute an important new area of medical research. hES cells have a demonstrated potential to be propagated in the undifferentiated state and then to be induced subsequently to differentiate into any and all of the cell types in the human body, including complex tissues. This has led to the suggestion that many diseases resulting from the dysfunction of cells may be amenab...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/0735C12N5/078C12N5/079C12N5/0793C12N5/077
CPCC12N5/0606C12N5/0619C12N5/0657C12N2506/02C12N5/0634C12N2501/998C12N2502/02C12N5/0621A01K67/0273A01K67/0275A01K2227/105A01K2267/0393C07K14/43595C12N5/0604C12N2501/235C12N2517/04C12N2533/52A61P1/04A61P1/16A61P13/00A61P13/12A61P17/02A61P19/08A61P21/02A61P21/04A61P25/00A61P25/16A61P25/28A61P3/10A61P35/00A61P3/06A61P35/02A61P37/04A61P9/00C12N5/16C12N5/0603
Inventor LANZA, ROBERT P.CHUNG, YOUNG GIE
Owner ADVANCED CELL TECH INC
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